Multiple Toxin Production in the Cyanobacterium Microcystis: Isolation of the Toxic Protease Inhibitor Cyanopeptolin 1020

Gademann, K; Portmann, C; Blom, J F; Zeder, M; Jüttner, F (2010). Multiple toxin production in the cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020. Journal of Natural Products, 73(5):980-984. Postprint available at: http://www.zora.uzh.ch

University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.uzh.ch

Originally published at: Gademann, K; Portmann, C; Blom, J F; Zeder, M; Jüttner, F (2010). Multiple toxin production in the Winterthurerstr. 190 cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020. Journal of Natural CH-8057 Zurich Products, 73(5):980-984. http://www.zora.uzh.ch

Year: 2010

Multiple toxin production in the cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020

Gademann, K; Portmann, C; Blom, J F; Zeder, M; Jüttner, F

Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: Gademann, K; Portmann, C; Blom, J F; Zeder, M; Jüttner, F (2010). Multiple toxin production in the cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020. Journal of Natural Products, 73(5):980-984. Multiple toxin production in the cyanobacterium microcystis: isolation of the toxic protease inhibitor cyanopeptolin 1020

Abstract

The isolation and structure of cyanopeptolin 1020 (hexanoic acid-Glu-N[-O-Thr-Arg-Ahp-Phe-N-Me-Tyr-Val-]) from a Microcystis strain is reported. Very potent picomolar trypsin inhibition (IC(50) = 670 pM) and low nanomolar values against human kallikrein (4.5 nM) and factor XIa (3.9 nM) have been determined for cyanopeptolin 1020. For plasmin and chymotrypsin, low micromolar concentrations were necessary for 50% inhibition. Cyanopeptolin 1020 was found to be toxic against the freshwater crustacean Thamnocephalus platyurus (LC(50) = 8.8 microM), which is in the same range as some of the well-known microcystins. These data support the hypothesis that cyanopeptolins can be considered as a second class of toxins in addition to the well-established microcystins in Microcystis. 1

Multiple Toxin Production in the Cyanobacterium Microcystis: Isolation of the

Toxic Protease Inhibitor Cyanopeptolin 1020

Karl Gademann†,‡,*, Cyril Portmann, ‡ Judith F. Blom,§ Michael Zeder, § and

Friedrich Jüttner§

Department of Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056 Basel,

Switzerland

Chemical Synthesis Laboratory, SB-ISIC-LSYNC, Swiss Federal Institute of

Technology (EPFL),

CH-1015 Lausanne, Switzerland

Limnological Station, Institute of Plant Biology, University of Zürich,

CH-8802 Kilchberg, Switzerland

* To whom correspondence should be addressed. Tel: ++41 61 267 11 44. Fax: ++41

61 267 11 45. E-mail: [email protected]

† University of Basel

‡ Swiss Federal Institute of Technology

§ University of Zürich

The isolation and structure of cyanopeptolin 1020 (hexanoic acid-Glu-N[–O–Thr-

Arg-Ahp-Phe-N-Me-Tyr-Val-]) from a Microcystis strain is reported. Very potent

picomolar trypsin inhibition (IC50 = 670 pM) and low nanomolar values against human kallikrein (4.5 nM) and factor XIa (3.9 nM) have been determined for

cyanopeptolin 1020. For plasmin and chymotrypsin, low micromolar concentrations

were necessary for 50% inhibition. Cyanopeptolin 1020 was found to be toxic against

the freshwater crustacean Thamnocephalus platyrus (LC50 = 8.8 µM), which is in the same range as some of the well-known microcystins. These data support the hypothesis that cyanopeptolins can be considered as a second class of toxins in addition to the well-established microcystins in Microcystis.

Cyclic depsipeptides containing the structural element 3-amino-6-hydroxy-2-

piperidone (AHP) (so-called cyanopeptolins) are widely distributed secondary metabolites in cyanobacteria.1 The structural diversity of previously described

compounds is large, but all can be considered inhibitors of serine proteases of

crustaceans and mammals.1 Although preferentially mammalian enzymes in the

context of pharmacological research have been tested, the evolutionary primary target

enzymes can be assumed to be those of aquatic invertebrates that are important

grazers of these cyanobacteria.1 Digestive serine proteases of these invertebrates are

strongly inhibited2 and could thus render the cyanobacteria unsuitable food for them.

The conserved feature of serine proteases in evolution allows these inhibitors to be

successfully applied also against mammalian enzymes. Most of the AHP-containing

compounds are active in the micromolar range,1c but some fit so perfectly in the

reaction center of mammalian serine proteases that nanomolar concentrations were

shown to be sufficient for inhibition.3 Co-crystallization experiments have been

conducted to analyze in atomic detail the binding properties of such an inhibitor to the

reaction center of mammalian trypsin.4 While the inhibition of digestive serine

proteases of various organisms is well established, on the acute toxicity of some of

these congeners is not understood. Microcystilide A when applied intraperitoneally to

mice induced convulsions and spasms but not death.5 Cyanopeptolin SS isolated from

a natural cyanobacterial bloom caused death of crustaceans6 as did oscillapeptin J for

Thamnocephalus platyurus.7

While little is known about the toxicity of cyanopeptolins, the most studied

cyanobacterial toxins are the microcystins, which exert their hepatotoxicity through

protein phosphatase inhibition8 and are produced by species of the genera

Microcystis, Anabaena, Oscillatoria, Planktothrix, and Nostoc. Microcystins are 4

suspected of countless animal poisonings throughout the globe and also thought to be

responsible for a severe incident in Brazil, where several persons died of

contaminated water.9 As a direct consequence, the World Health Organization set guidelines for drinking water and defined threshold values for microcystin concentration.9 However, cyanobacteria of the genus Microcystis are known to

produce several classes of compounds in addition to the microcystins frequently

totaling more than 10 distinct secondary metabolites. For example, Microcystis

aeruginosa PCC 7806 is known to produce cyanopeptolins A-D, cyanopeptolin 963A,

[D-Asp3]microcystin-LR and microcystin-LR, and the aerucyclamides A-D.10 While

most of the attention on toxic cyanobacterial peptides focused on the microcystins, the above mentioned reports on toxic effects of cyanobacteria might imply the presence

of additional toxins related to cyanopeptolins in Microcystis. Thus, one could propose

that potent and unselective trypsin inhibitors are able to shut down other processes

than protein digestion by virtue of their binding to other serine proteases.

Here we report on an extremely potent, picomolar trypsin inhibitor, cyanopeptolin

1020 (1), which also binds in the low nanomolar range to factor XIa and human

kalllikrein. In addition, this compound exhibits acute toxic properties against the

crustacean Thamnocephalus platyurus. In addition to several microcystins found in

the producing strain, cyanopeptolin 1020 (1) constitutes a second toxic compound

class, which thus provides supporting evidence for multiple toxin production in

Microcystis.

NH O 2 OH

H2N N N N H H O NH O O H N O N CH3 O H O O N N CH3 H O O O

OH Cyanopeptolin 1020 (1)

CH CO2H 3 O N HN NH CH3 CH3 H3C O CH2 O O O NH HN H H H CH3 CH3 N N O CH3 O CO2H O H3C CH3

Microcystin-LA

The 60% methanolic extract of the biomass of Microcystis aeruginosa UV-006 was fractionated and assayed against the sensitive freshwater crustacean Thamnocephalus platyurus for acute toxicity. Overall, more than 20 different compounds were present in these fractions, and out of totally 16 fractions, nine were found to display acute toxicity in this assay. Among these nine fractions, eight compounds were assigned as known or new microcystins (Table S1, see supporting information). So far microcystins of all Microcystis strains have been shown to contain the structural element N-methyldehydroalanine rather than the isomeric dehydrobutyrine. Under this presumption, we found the very common and typical microcystin congeners

[Asp3]-MC-LR and MC-LR. MC-LR and MC-LA have already been described in a previous study on this strain11 and we could confirm both components. In addition, we 6

found the lipophilic congeners MC-LAba, MC-LA, MC-LV and MC-LL which were

only rarely reported before.12 Additionally, two new variants were found (assigned

based on typical absorption and mass spectra), which have not been described yet (see

table S1, supporting information). However, the amounts available were too small to

allow the determination of the structure.

Interestingly, one compound displayed similar toxicity as MC-LA, but based on the

UV spectrum, it was hypothesized that this compound is not a microcystin, but a

structurally different peptide. In order to elucidate the structure of this toxin, this

fraction was purified and analyzed by NMR spectroscopy and chemical degradation

(amino acid analysis). The molecular formula of 1 was determined to be

C50H72N10O13 based on HRMS complemented by isotope labeling studies that

established the presence of 10 N atoms. This molecular formula was consistent with

the NMR data (Table 1). The 1H NMR spectrum displayed the typical pattern of a

peptide. The different amino acids were identified based on 1H,1H COSY, 1D-

TOCSY and HMBC data as Glu, Thr, Arg, Ahp, Phe, N-Me-Tyr, Val and hexanoic

acid (HA), see also the supporting information for full details. The sequence was

obtained by 1H-13C long-range correlations: From Glu-NH to HA-C1, from Thr-NH to Glu-C1, from Arg-NH to Thr-C1, from Thr-H3 to Val-C1, from Val-NH to N-Me-

Tyr-C1, from N-Me-Tyr-NCH3 to Phe-C1 and from Ahp-NH to Arg-C1. The

macrocyclic ring was established by a ROESY interaction between Ahp-H5 and Phe-

H3a, and Ahp-NH and Arg-H2 (Figure 1). This sequence assignment was

corroborated by elaborate MS experiments of both natural abundance and 15N enriched peptides. The mass fragmentation pattern of cyanopeptolin 1020 (1) has been found previously in complex mixtures by MALDI-TOF MS. 2b The fragment ion

at m/z = 904 results from the loss of the hexanoyl fragment (Hex) and water. 7

Moreover, the detection of a peak at m/z = 794 (containing 9 N atoms) is corresponding to the loss of the Hex-Glu side chain. The fragment ion at m/z =

467 (6 N atoms) was assigned to the fragment Hex-Glu-dehydrobutyrate-Arg resulting from cleavage of the peptide bond C-terminal at Arg and dehydration of the

Thr residue. The complementary fragment Ahp-Phe-N-Me-Tyr-Val-OH(-H2O) (m/z =

534) could also be detected. The mass detected at m/z = 420 (containing 3 N atoms) was assigned to the Phe-N-Me-Tyr-Val-OH (-H2O) fragment. Based on this evidence resulted from MS and NMR experiments, we assign the constitution as shown for 1 to cyanopeptolin 1020.

Figure 1. Key COSY, ROESY and HMBC correlations in cyanopeptolin 1020 (1).

The configuration of 1 was determined by hydrolysis and the enantiomers were separated following Marfey’s method. This procedure established the configuration of the amino acids as L-Glu, L-Thr, L-Val, L-N-Me-Tyr, L-Phe and L-Arg. For the Ahp residue, the relative configuration was assigned based on ROESY interactions between Ahp-NH and Ahp-H3a, Ahp-H3a and Ahp-OH, Ahp-H2 and Ahp-H3b, Ahp- 8

H2 and Ahp-4a. Therefore, the OH group is positioned in a pseudo axial position,

which can be explained by the anomeric effect. The absolute configuration of the Ahp

residue was established by oxidation with CrO3, followed by hydrolysis and Marfey’s

derivatization to yield L-Glu.

Cyanopeptolin 1020 (1) belongs to a large group of related cyclodepsipeptides that

feature the Ahp residue as a key structural unit.1 These peptides have been described

in the literature as protease inhibitors, where basic amino acids adjacent to Ahp such as Arg or Lys convey selectivity for trypsin, and hydrophobic amino acids result in

chymotrypsin selectivity. We have evaluated the inhibitory activity of cyanopeptolin

1020 for trypsin, chymotrypsin and several other proteases involved in blood

coagulation (Table S2, supporting information). Cyanopeptolin 1020 inhibited trypsin

with an IC50 value of 0.67 nM. This potent inhibitory power against trypsin is

remarkable, and it represents one of the strongest and most potent Ahp-containing

3 inhibitors that have been isolated to date. When compared to A90720A (IC50 = 10 nM against bovine trypsin), cyanopeptolin 1020 (1) features a Phe residue instead of a

Val, and the side chain D-Leu/sulfated glycerate is replaced by a L-Glu/hexanoic acid

combination. Based on the crystal structure of the A90720A/bovine trypsin complex

(PDB code 1TPS),4 we docked cyanopeptolin 1020 into the active site of bovine

trypsin (AutoDock 4.0).13 Conformational searching followed by docking converged

to the general conformation shown in Figure 2 (Cyanopeptolin 1020, green;

A90720A, grey). The basic interactions for 1 with bovine trypsin match the one found in the crystal structure: the Arg side-chain recognizes the P1 binding site, and the Ahp

residue modulates the conformation of the side chains of the catalytic triad Asp-His-

Ser. It has been suggested by structural work by two groups3,14 that Ahp-containing

inhibitors prevent a water molecule from being close to the scissile bond. The sulfated 9

glycerate residue is replaced by Glu in 1 and the Phe residue occupies the same

position as Val in the 1TPS structure. However, the presence of the bulky Phe residue

in lieu of Val forces the N-Me-Tyr residue inward, which might result in a more

favorable interaction with trypsin, and thus in increased binding.

Figure 2. Crystal structure of A90720A (grey) /bovine trypsin complex (PDB code 1TPS) with cyanopeptolin 1020 (1, green) docked into the active site.

In addition to the IC50 value of depsipeptide 1 against trypsin in the picomolar range, low nanomolar IC50 values against factor XIa (3.9 nM) and kallikrein (4.5 nM)

were determined. For plasmin and chymotrypsin, IC50 values of 0.49 and 1.8 M, respectively, could be measured. We did not find inhibitory properties against the human plasminogen activator, thrombin and human LMW urokinase at concentrations below 2.5 µM. Although these values cannot be directly compared with each other, as different enzymes, different substrates and different concentrations have been used in the experiments, generally very potent inhibition values have been used. Therefore, 10

these values confirm that the cyanopeptolin 1020 (1) is a potent inhibitor of a series of serine proteases.

Additionally, we wanted to confirm the toxicity of cyanopeptolin 1020 (1) against the sensitive freshwater crustacean Thamnocephalus platyurus using the highly purified cyanopeptolin 1020 (1). T. platyurus serves as a benchmark organism for the evaluation of the ecotoxicological potential of natural products.15 We have determined

an LC50 value of 8.8 M in the 24-h acute toxicity assay (Figure S14, supporting

information). This value is comparable to those determined for microcystins, which

16 represent the prototype example of cyanobacterial toxins. For example, LC50 values

of 10.8, 4.7 and 7.0 M have been measured for MC-LR, MC-YR and MC-RR,

16 respectively. The low LC50 value of cyanopeptolin 1020 (1) thus clearly establishes

that this compound constitutes another group of toxins in Microcystis of similar

potency as several microcystins. Such toxicity might also be unveiled for other

cyanopeptolins, and one related natural product from Planktothrix rubescens,

oscillapeptin J, has been described of similar acute toxic activity.7 It can be speculated

that these toxic effects are related to the very potent serine protease inhibition as

described above, in particular as the large family of trypsin-like proteases17 are involved in many key processes related to the viability of organisms. More detailed toxicological studies on mammals are thus encouraged by this study.

In this note, we have reported the structure elucidation of cyanopeptolin 1020 (1) extracted from Microcystis aeruginosa UV-006, which inhibits trypsin in the picomolar range and factor XIa and kallikrein in the low nanomolar range. This compound was shown to be toxic to the freshwater crustacean T. platyurus, and its toxicity was determined to be comparable to commonly found microcystins, which are frequently described as biotoxins from cyanobacteria. This report thus suggests 11

the presence of multiple toxins in Microcystis aeruginosa UV-006 and encourages further evaluation of toxicity of cyanopeptolin 1020 (1) in mammals. The co- occurrence of toxic cyanopeptolin 1020 (1) along with several microcystins in

Microcystis might also suggest a re-evaluation of drinking water guidelines and public health concerns currently solely focusing on microcystins to include other relevant toxins such as the presented cyanopeptolins as well. 12

Experimental Section

General Experimental Procedures. NMR spectra were acquired on a Bruker

DRX-600 equipped with a cryoprobe and are referenced to residual solvent proton

and carbon signals (H 2.50, c 39.5 for DMSO-d6). Accurate mass ESI spectra were

recorded on a Bruker FTMS 4.7T BioAPEX II at the University of Fribourg. The

identification and the purification were performed on a HPLC Shimadzu 10AVP

system equipped with a photodiode array detector RF-10AXL and an ESI mass

spectrometric detector LCQ Duo (Finnigan Thermo). HPLC analyses for Marfey’s

experiments were performed on a Dionex HPLC system equipped with a P680 pump,

an ASI-100 automated sample injector, a TCC-100 thermostated column

compartment, a PDA-100 photodiode array detector and a MSQ-ESI mass spectrometric detector.

Culture, Extraction and Isolation. The Microcystis aeruginosa strain UV-006

was an isolate from Hartebeespoort Dam near Pretoria, South Africa.11 M. aeruginosa

UV-006 was grown in mineral medium at 26 °C under constant light conditions (40 ±

5 µmol m-2 s-1). Glass tubes with a diameter of 7 cm and a height of 1.5 m containing

5 L media were used. The tubes were aerated with 200 mL min-1 compressed air

containing 1000 ppm CO2 and continuously stirred with a magnetic stirring bar. Cell

biomass was harvested using a flow-through centrifuge (Westfalla Separator AG,

Germany) and subsequently stored at -23 °C. To obtain high percentage labelled

15 - cyanopeptolin 1020 (1), nitrate was replaced by NO3 in the medium (final

concentration 4.0 mM).

Cyanopeptolin 1020 (1) was extracted by the addition of 10 ml 60% methanol per g

of wet cell biomass, overnight incubation at 1 °C in the dark and centrifugation for 10

min at 27500 × g. Compounds in the supernatant were brought to dryness in a rotatory 13

evaporator, dissolved in 2 ml 60% MeOH and filtered (0.2 µm pore size PTFE syringe

filter). The solvent was removed under a moderate stream of nitrogen and the mixture

was stored in glass vials at -23 °C. Compound 1 was then purified by HPLC with a

reversed-phase column (C-18 Grom-Sil 120 ODS-4 HE, 250 x 4.6 mm, 5 µm particle

size, Stagroma) with a flow rate of 1 ml min-1 and a column temperature of 30 °C.

Separation and identification were performed with a binary solvent gradient of UV-

treated deionised H2O (solvent A) and CH3CN (solvent B), both acidified with 0.05%

TFA (trifluoroacetic acid). The following gradient was used: 0 min: 30% B; 10 min:

35% B; 40 min: 70% B, 42 min: 100% B; 50 min: 100% B; compound 1 eluted after

13.0 min. TFA was removed before evaporating the solvents by applying sequentially

the combined HPLC fraction on a C18 SPE cartridge (5 g, conditioned with 10%

MeOH). The cartridge was then washed with H2O to remove TFA and 1 was finally

eluted with 100% MeOH. Subsequently, the MeOH was removed under reduced

pressure and H2O was removed by lyophilisation to afford 1 as a colourless amorphous solid.

Cyanopeptolin 1020 (1): UV (47% acetonitrile in water with 0.05% TFA) max 277

1 13 + nm; H and C NMR data (DMSO-d6), see Table 1; HRESIFTMS m/z [M+H]

1021.5343 (calcd for C50H73N10O13, 1021.5353)

Marfey’s Analysis. Compound 1 (0.1 mg, 0.1 mol) was hydrolyzed in a sealed tube with 6 N HCl (0.3 mL) at 105 °C for 24 h. The solution was concentrated to dryness with a stream of N2. The hydrolyzed compound was treated with 60 L of a solution of 1% N-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA) in acetone

and 60 L of 6% triethylamine in a sealed vial at 50 °C for 1 h. To the mixture 60 L

of 5% acetic acid were added and the mixture was dried under a moderate stream of 14

N2. The residue was dissolved in 200 L of MeOH and analyzed by RP-HPLC using an Agilent Zorbax SB-C18 column (150 x 2.1 mm, 3.55 m). Mobile phase A was

5% acetic acid in H2O, B was CH3CN / MeOH (9/1) and C was CH3CN. The linear

gradient started with 5% B to reach 50% B over 50 min. The column was then washed

for 10 min with 100% B, followed by 20 min with 100% C. The column was

stabilized before the next injection with 5% B for 20 min and maintained at 50 °C.

The flow rate was set at 0.2 mL / min. The configuration was determined by

comparison with the retention time of derivatives from commercially available amino acids. The retention time for each amino acid derivative was obtained by extracting the mass of the derivative in the HPLC-MS chromatogram. Retention times (min) for the standard amino acid derivatives were determined as follows: L-Thr 25.2; L-allo-

Thr 26.4; D-Thr 32.3; D-allo-Thr 29.5; L-Arg 21.6; D-Arg 22.3; L-Phe 44.8; D-Phe

50.1; L-Val 40.5; D-Val 46.7; L-N-Me-Tyr (di-FDAA derivative) 53.7; D-N-Me-Tyr

(di-FDAA derivative) 54.4; L-Glu 27.6; D-Glu 30.8. The configuration of

cyanopeptolin 1020 (1) was unambiguously assigned as L-Thr 24.9; L-Arg 21.6; L-

Phe 44.9; L-Val 40.0; L-N-Me-Tyr (di-FDAA derivative) 53.7 and L-Glu 27.6; see

also the Supporting Information for chromatograms.

Configuration of Ahp. The oxidation was realized according to the procedure

developed by Itou et al.18 Cyanopeptolin 1020 (1 , 0.1 mg, 0.1 mol) was oxidized in a solution (0.5 mL) of CrO3 in AcOH (1 mg / mL) at room temperature for 2 hours.

The mixture was then applied onto a C18 SPE cartridge conditioned with 10% MeOH.

After applying the mixture, the cartridge was first washed with H2O, and the material was eluted with MeOH. After evaporation of the MeOH the resulting oxidized material was hydrolyzed, derivatized and analyzed as described above. Only the

Marfey’s derivative of L-Glu was observed, thus establishing the configuration of 15 position 2 of the Ahp residue to be S; see also Supporting Information for chromatograms.

Acute Toxicity Assay. The highly purified cyanopeptolin 1020 (1) was tested in a

24 h acute toxicity assay with Thamnocephalus platyurus (Thamnotoxkit FTM;

MicroBioTests Inc.) in a multiwell plate using instar II-III larvae hatched from cysts.

The cyanopeptolin was tested in six concentrations ranging from 0.1 to 100 µM. For every concentration, 20 to 30 animals have been used. The nonlinear regression analysis and determination of the LC50 value were calculated with Graph Pad Prism 4 for Windows.

Protease Inhibition Assays. The details of the proteases and their respective fluorogenic substrates are summarized in Table S2. The enzyme inhibition assays were carried out in using modified literature procedures.19 The kinetic fluorescence measurements were realized with transparent microplates (Nunc) using a fluorescence microplate reader (Tecan infinite M1000) set at em = 355 nm, ex = 480 nm and measuring a datapoint every 30 seconds for 1 h. To obtain the initial reaction rate, linear regression was performed with Microsoft Excel. The enzymes were dissolved in a buffer containing 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1 mM

CaCl2, 0.1% BSA and 0.01% Triton X-100. To facilitate the dissolution of the substrates, 7.5% DMSO was added. The DMSO amended buffer was also used as blank. Optimal enzyme concentrations were determined by measuring kinetics with different enzyme concentration. The substrate solution (50 µL) was added to a mixture of 50 µL of the enzyme solution and 50 µL of the blank. The enzyme concentrations were assayed between 0.1 and 10 nM (final concentration). The optimal protease concentrations are listed in Table S2. For the inhibition assays, a 16 mixture of 50 µL of protease solution and 50 µL of inhibitor solution were incubated at RT for 30 min. The substrate solution (50 µL) was then added and the measurement was directly started. The fractional activity was obtained by dividing the inhibited rate by the uninhibited rate obtained with a blank reaction. Chymotrypsin inhibition assays were performed as stated recently.7 The inhibition curves and the 50% inhibition values (IC50) were calculated using the non-linear regression sigmoidal four parameter formula with Prism 5.0b for Mac OS X.

Acknowledgment

K.G. is a European Young Investigator (EURYI) and thanks the SNF for financial support (PE002-117136/1). M.Z. was supported by the Hydrobiologie-Limnologie-

Stiftung für Gewässerforschung. We thank Professor C. Heinis (EPFL) for helpful discussions and technical help with some of the protease assays.

Supporting Information Available. Detailed structure elucidation, 1H-NMR,

COSY, ROESY, HSQC and HMBC spectra, chromatograms and data of the enzyme assays are provided as supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

Table 1. NMR Spectroscopic Data (600 MHz, DMSO-d6) for Cyanopeptolin 1020 (1).

c Residu Posit C H (J in Hz) HMBC ROESY e ion

HA 1 172.5, C

2 34.7, CH2 2.13, m HA-1, HA-3, HA-4

a 3 24.6, CH2 1.51, m HA-1, HA-2, HA-4, HA-5 HA-4

4 30.5, CH2 1.23, m HA-5

5 21.6, CH2 1.27, m HA-4, HA-6

6 13.6, CH3 0.85, t (7.0, 7.0) HA-4, HA-5

Glu 1 172.1, C

2 51.5, CH 4.39, ddd (9.1, 7.8, Glu-1, Glu-3 Glu-3b, Glu-4 5.1)

b 3a 26.7, CH2 1.88, m

3b 1.73, m

4 30.0, CH2 2.26, m Glu-3, Glu-5 Glu-3a

5 173.9, C

NH 8.02, d (7.8) HA-1, Glu-2 Glu-2, Glu-3b, Glu-4, HA-2

Thr 1 168.8, C

2 54.3, CH 4.57, d (9.3) Glu-1, Thr-1 Thr-4

3 71.7, CH 5.42, m Val-1, Thr-4 Thr-4

4 17.5, CH3 1.16, d (6.6) Thr-2, Thr-3

NH 7.85, d (9.3) Glu-1 Thr-2, Thr-4, Glu-2

Arg 1 169.4, C

2 51.1, CH 4.23, m Arg-3a

b 3a 27.0, CH2 1.88, m Arg-3b

3b 1.38, m

a 4 24.6, CH2 1.38, m

5 39.9, CH2 3.05, m

6 156.4, C

NH 8.49, d (8.9) Thr-1 Ahp-NH, Thr-2, Thr-3

NH2 7.46, m Arg-5

Ahp 1 168.6, C

2 48.4, CH 3.62, m Ahp-1 Ahp-3b, Ahp-3a, Ahp-4a

3a 21.3, CH2 2.40, m Ahp-3b

3b 1.60, m 18

4a 29.0, CH2 1.68, m

4b 1.56, m

5 73.5, CH 5.07, m Ahp-1 Ahp-4a, Ahp-4b, Phe-3a

NH 7.11, d (9.2) Arg-1 Ahp-2, Ahp-3a, Thr-3, Arg-2

OH 6.05, d (3.0) Ahp-3a, Ahp-4a, Ahp-5, Val-5

Phe 1 170.1, C

2 50.0, CH 4.75, dd (11.8, 3.7) Phe-1 Phe-3b

3a 35.0, CH2 2.90, dd (13.9, 11.8) Phe-1’, Phe-2’/6’ Phe-3b

3b 1.80, dd (13.9, 3.7) Phe-2’/6’

1’ 136.5, C

2’/6’ 129.2, CH 6.84, d (7.3) Phe-3, Phe-2’/6’, Phe-5’ Phe-2, Phe-3a, Phe-3b, Ahp-4b,Ahp-2, Ahp-5

3’/5’ 127.5, CH 7.18, dd (7.3, 7.3) Phe-2’/6’, Phe-3’/5’, Phe-4’ Phe-2’/6’, N-Me-Tyr-2’/6’, N-Me-Tyr-3’/5’

4’ 125.9, CH 7.14, d (7.3) Phe-2’/6’

N-Me- 1 169.0, C Tyr

2 60.7, CH 4.89, dd (11.6, 2.3) N-Me-Tyr-1 N-Me-Tyr-3a, Phe-2

3a 32.5, CH2 3.11, dd (14.2, 2.3) N-Me-Tyr-1’, N-Me-Tyr-2’/6’ N-Me-Tyr-3b

3b 2.72, dd (14.1, 11.6) N-Me-Tyr-1’, N-Me-Tyr-2’/6’

1’ 127.3, C

2’/6’ 130.1, CH 7.00, d (8.4) N-Me-Tyr-3, N-Me-Tyr-2’/6’, N-Me-Tyr-2, N-Me-Tyr-3’/5’, Phe-2, Phe-3a, N-Me-Tyr-4’ Phe-3b

3’/5’ 115.1, CH 6.78, d (8.4) N-Me-Tyr-1’, N-Me-Tyr-3’/5’, Ahp-OH (really weak) N-Me-Tyr-4’

4’ 156.0, C

NCH 30.0, CH3 2.76, s Phe-1, N-Me-Tyr-2 3

OH 9.34, s N-Me-Tyr-3’/5’, N-Me-Tyr-4’ N-Me-Tyr-3’/5’

Val 1 171.8, C

2 55.5, CH 4.71, dd (9.6, 4.5) Val-1, Val-3, Val-5, N-Me- Val-3, Val-4 Tyr-1

3 30.4, CH 2.07, m Val-4, Val-5

4 18.9, CH3 0.87, d (6.8) Val-2, Val-3, Val-5

5 16.9, CH3 0.72, d (6.8) Val-2, Val-3, Val-4

NH 7.43, d (9.6) N-Me-Tyr-1 Val-2, Val-5, N-Me-Tyr-2, N-Me-Tyr-NCH3, Ahp-OH

a b these two carbons can be interchanged,

c HMBC correlations are given from proton(s) stated to the indicated carbon atom.

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